Apoptosis is a fundamental biological mechanism of the cell, by which it executes its own death upon receiving appropriate signals. Our long-term objective is to understand the molecular mechanisms of this process to develop effective therapeutic strategies for a number of pathological conditions including cancer, neurodegenerative diseases, and aging, where apoptosis plays a key role. Death signals appear to impinge on mitochondria in many cases, resulting in the release of key effector proteins from the intermembrane space. We have shown that Bcl-2 family proteins permeabilize the outer membrane directly, using in vitro systems such as outer membrane vesicles and cardiolipin containing liposomes. Even though 2000 kDa dextrans are released from these vesicles by pro-apoptotic Bcl-2 family members, Bax and Bid, morphological changes in the membranes are not detectable by conventional EM. Based on this observation, we hypothesize that changes occurring during membrane permeabilization are transient. We will therefore use high magnification and resolution imaging technology that can capture transient changes. The specific aims are to: 1. Apply atomic force microscopy to image changes in the membrane induced by Bax and Bid as well as to visualize Bax oligomers. We will deposit liposomes and outer membrane vesicles on mica to scan the surface after the addition of Bax and Bid. We will monitor the changes in real time. 2. Apply cryo-EM and freeze-fracture to visualize the lipid bilayer and the outer membrane and detect changes before and after the addition of Bax and Bid. Both liposomes and OMVs will be frozen at various times to capture changes. [unreadable] [unreadable]